Hypertrophic cardiomyopathy (HCM), a genetic-based disease of the heart and a leading cause of sudden death in young adults and competitive athletes, has been linked to mutations in key elements of the cardiac sarcomere including cardiac troponin T (cTnT) and alpha tropomyosin (alphaTm). The focus of this proposal is to determine the structure/function relationships of mutant cTnT and alphaTm proteins in cardiac myocytes. Studies on mutant TnT and Tms are important because: a) these are essential proteins of the thin filament regulatory system, b) mutant TnT and alphaTm proteins appear to be particularly malignant because they are associated with significant cardiac disease as manifested by a high incidence of sudden death, c) there is a large gap in knowledge regarding how specific mutations of these molecules lead to the disease state in cardiac muscle, and d) for the mutant Tm genes, which are expressed in numerous cell types in the body including skeletal muscle, it is presently unclear why disease appears to be restricted to cardiac muscle. The specific aims are: 1 . Test the hypothesis that mutant TnTs will incorporate normally into the sarcomere, without alterations in the stoichiometry of the contractile apparatus or in the sarcomeric architecture of the adult cardiac myocyte. It is hypothesized that the mutant TnTs will exert their action by directly disrupting Ca2+-activated contractile function, including force development and shortening velocity. 2. Test the hypothesis that mutant Tms will incorporate normally into the sarcomere in the absence of alterations in the stoichiometry of the contractile apparatus or in sarcomeric architecture of the adult cardiac myocyte. It is hypothesized that the mutant Tms will directly cause alterations in Ca2 +-activated tension generation. In addition, it is hypothesized that the ectopic expression of the skeletal muscle isoforms of TnT or TnI will lessen contractile dysfunction by mutant Tms in cardiac myocytes, and account for the apparent cardiac-lineage restriction for dysfunction of the mutant Tms. The experimental strategy involves the rapid and efficient transfer of myofilament genes into adult cardiac myocytes in primary culture using recombinant adenovirus vectors. By combining myofilament gene transfer with single cardiac myocyte functional studies this proposal is uniquely positioned to provide the timely and comprehensive analysis of TnT and alphaTm structure/function to delineate the primary mechanism by which these mutant sarcomeric proteins cause HCM.